Project Summary/Abstract Seasonal influenza has an annual global incidence of up to 10% in adults and up to 30% in children. About 3 to 5 million infections result in severe disease while somewhere between a quarter and a half million cases are fatal. Antiviral drugs can lessen the severity and reduce mortality when administered within 48 h of onset of symptoms. Hence, sensitive and specific point-of-care (POC) diagnostic tools, especially those that can be used at home, can be invaluable in expediting appropriate therapy and mitigating viral spread. While TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR) and viral culture continue to be the diagnostic gold standards, several rapid POC influenza diagnostic technologies have been commercialized. Although results are available within 15 min, most of these tests have poor sensitivity (40-70% compared to gold standards). Rapid tests based on isothermal nucleic acid amplification (Alere i influenza A and B test) have also recently become available, and while they are very specific and sensitive, they can be too expensive for widespread POC use - $70 to $85 per test, and requiring the Alere i instrument that itself costs about $8500. To reduce testing cost and make nucleic acid diagnostics more widely available for at- home testing we propose to develop a one-pot, robust, and cheap POC test that can distinguish influenza A and B viruses. Key to this technology is viral RNA amplification by loop-mediated isothermal amplification (LAMP) and conversion of the resulting nucleic acid amplicons into glucose for readout via widely available, over-the-counter glucometers. Overall, in a partnership between Paratus Diagnostics and the Ellington lab at the University of Texas at Austin, we will develop a Paratus Consumable Cartridge (PCC) that will serve as an all-in-one device that extracts, amplifies, and transduces pathogen genetic signatures from patient samples to glucometers for readout, and is projected to cost about $5/device. In particular, we will develop reverse transcription (RT)-LAMP assays specific for the influenza A and B virus matrix genes (Aim 1, Objective 1) and transduce the amplicons into glucose via our novel, patent-pending sequence-specific oligonucleotide strand displacement (OSD) reporters. (Aim 1, Objective 2). We will automate the molecular assay on the PCC (Aim 2, Objective 3) and using biological samples perfect field-appropriate sample extraction procedures requiring only intuitive user input (Aim 2, Objective 4). Diagnostic utility of the PCC and the molecular assay will be demonstrated by comparing its robustness and its positive and negative predictive values with those of TaqMan real-time RT-PCR when challenged with blinded surrogate clinical samples (Aim 2, Objective 5). These data will position the PCC-based POC diagnostic for further validation with clinical samples and eventually field trials.